Refractory Leukemia Research Articles

Impact of Outpatient Administration of Cytarabine Consolidation Chemotherapy on Hospital Length of Stay in Adults with Acute Myeloid Leukemia

Background: Administration of 3-4 cycles of high/intermediate dose cytarabine (H/IDAC) reduces relapse of acute myeloid leukemia (AML) and bridges patients (pts) to allograft. There is no accepted standard model of safely delivering H/IDAC in these pts. To reduce reliance on inpatient (inpt) chemotherapy administration and safely avoid or forestall hospitalization during cycles, we developed an outpatient (outpt) protocol to deliver H/IDAC. Pts were assigned outpt or inpt treatment at their hematologist's discretion. We aimed to evaluate the benefits and safety of our model in this retrospective observational study. Methods: Pts diagnosed with AML and treated with intensive induction chemotherapy between 2015 and 2020 were identified; included pts received ≥1 consolidation cycle of 1-3g/m² cytarabine over 3 hours BID, dose adjusted for age and renal function, on days 1, 3 & 5 of a planned 28-day cycle. Outpts received H/IDAC via computerized ambulatory delivery device (CADD) pump and central venous access device (CVAD), attended days 1-6, then 3 times a week for blood product support until count recovery; elective readmission was not mandated. Pts were educated to present to hospital if unwell. Inpts were admitted for H/IDAC but could be discharged prior to count recovery. Antimicrobial prophylaxis consisted of antifungal agents and valaciclovir. The primary endpoint was mean duration of hospital admission (LOS) per consolidation cycle. As pts may have been treated on an inpt and/or outpt basis, generalized estimating equations were used to compare LOS clustered by pt. Secondary outcomes included death during H/IDAC; intensive care unit (ICU) admission; time to admission; blood product use; cycle length; bacteremia; time to count recovery; relapse free survival (RFS) and overall survival (OS). Results: Of 109 pts identified, 62 pts were included and 47 pts excluded (not AML=3; induction death=7; refractory leukemia=14; alternative=13; no consolidation=10). Median age was 55.2 years (range 18.2-70.3); 31 (50%) males; ELN 2010 fav=23 (37.1%), Int-2/3 = 30 (48.4%), Adv=8 (12.9%); denovo AML in 60 (96.7%); and therapy related in 2 (3.2%). FLT3-ITD, NPM1 and bi-allelic CEBPA mutations detected in 11 (17.7%), 27 (43.5%) and 2 (3.2%) pts, respectively. Fifty-nine pts (95.1%) received 7+3 idarubicin induction and 7 (11.3%) received midostaurin. A total of 131 cycles of H/IDAC were administered; 81 (61.8%) were commenced as outpt and 50 (38.2%) as inpt. Twenty-seven (43.6%) pts commenced all cycles as an outpt. Those commencing consolidation as an outpt spent, on average, 10.3 fewer days in hospital per cycle. The mean (SD) LOS for outpt and inpt was 11 (8.3) versus 21.3 (8) days (p<0.001), respectively. For each consolidation cycle, outpts spent less time in hospital (mean, [SD]): cycle 1, 12.6 (10.3) vs 20.7 (8.0) days (RR 1.6, p=0.007); cycle 2, 11.6 (7.2) vs 21.4 (8.3) days (RR 1.8, p=0.001); cycle 3, 8.4 (5.8) vs 24.6 (7.4) days (RR 2.9, p<0.001). There were no deaths among inpts and one death in outpts (septic shock). ICU admission occurred in 8/81 (9.9%) cycles in outpts vs 1/50 cycles (2%) in inpts (p=0.12). Mean ICU LOS for outpts vs inpts in cycle 1 was 17.6 days vs 1.6; cycle 2, 5.1 vs 0 days; and cycle 3, 4.2 vs 0 days. Bacteremia was detected in 41 cycles (31.3%), with 38 isolates (gram pos=17, gram neg=21) in 31 outpt cycles (38.2%) and 15 isolates (gram pos=6, gram neg=9) in 10 inpt cycles (20%). Median time to admission in outpts was 14 days (IQR, 3-21); 15 (18.5%) cycles did not result in pt admission. There were no differences in timing of admissions by cycle number nor time-point to prompt elective admission (Fig.1). There was no difference in packed cell or platelet transfusion requirement between inpts and outpts. Time to blood count recovery was similar. No improvement was seen in cycle length. The proportion undergoing allogeneic transplant in first remission was 37.1%, and at any time, 59.7%. Over a median follow up of 3.3 years, median RFS was 2 years and OS, not reached. Conclusion: In selected pts, outpt based AML H/IDAC consolidation therapy significantly reduced hospital length of stay compared with inpts. There was no impact on cycle length. Bacteremia and ICU admission rates were higher in outpts, and strategies such as remote monitoring or pre-emptive hospitalization could be considered. Our findings suggest outpt consolidation should be considered in a supported clinical setting.

Deploying an RNA-Based Gene Writer System and Lipid Nanoparticle (LNP) Delivery to Generate Functional Chimeric Antigen Receptor (CAR) T Cells with in Vitro and In Vivo Anti-Tumor Activity

The FDA approval of autologous Chimeric Antigen Receptor (CAR) T-cell therapies have added a powerful tool in the armamentarium to treat relapsed and refractory leukemias and lymphomas. However, significant challenges limit these therapies from accommodating current patient demand including supply chain limitations and wait times associated with availability of GMP quality viral vectors and long needle-to-needle time from initiation of treatment to receiving the final drug product. As of yet other competitive therapies including bi-specific antibodies and allogeneic CAR-T have not achieved the same level of therapeutic efficacy as conventional autologous T cell therapies highlighting the critical unmet need for same day treatment options for autologous CAR-T products. RNA Gene Writers leverage target-primed reverse transcription (TPRT) biochemistry evolved from non-LTR retrotransposon mobile genetic elements to modify the genetic information in cells using RNA templates and without the need to introduce DNA breaks. Moreover, RNA Gene Writers can be engineered to catalyze a variety of editing reactions, such as the introduction of gene-length DNA sequences, substitutions, insertions, and deletions. These edits can be achieved with all-RNA delivery in primary cells and in vivo, eliminating the necessity for viral vectors and DNA template-based gene editing. LNP-RNA delivery of RNA Gene Writers resulted in the integration and expression of transgenes in >60% of primary human T cells in vitro. Furthermore, our LNPs deliver RNA to T cells in vivo, with 80% reporter expression in a humanized mouse model and 45% in non-human primates (NHP). We have demonstrated LNP delivery of RNA Writers into primary human T cells with an RNA template encoding a CAR cassette can achieve >20% CAR+ T cells without detriments to cell viability or proliferative capacity and have demonstrated ability to mediate tumor cell killing in vitro. Moreover, RNA Gene Writer derived CAR-T cells can be introduced into mouse xenograft models to clear antigen specific tumors in vivo. CAR mRNA can also be packaged into proprietary LNP formulations and delivered to primary human T cells in a mixture of lymphocytes commonly found in patient leukapheresis to generate CAR-T cells in vitro. This opens the possibility of using RNA Gene Writers to develop a same-day CAR-T treatment. In addition, the modularity of our RNA Gene Writing technology allows multiplex editing to co-introduce multiple genetic changes including generation of universal and more potent CAR-T cells through the knock-out of B2M and TRAC at comparable levels (>80% double knockout) to Cas9 nucleases. Further, we show that we can achieve both edits simultaneously in 80% of T cells that have achieved CAR transgene insertion via another RNA Gene Writer enzyme. Importantly, we have demonstrated successful B2M and TRAC double knock-out without inducing translocations (undetectable vs 8% translocations with Cas9). RNA Gene Writing technology is uniquely positioned for same-day delivery of all-RNA components toenable a wide range of editing applications, from gene knock-out to gene integration. The ability to package the RNA Gene Writer system into LNPs unlocks the potential for the genetic engineering of autologous T cell therapies to enable same-day delivery a current limitation of conventional lentiviral-based CAR-T therapies.

Acute Lymphocytic Leukemia (ALL) in Pregnancy, Case Report and Literature Review

Introduction: Leukemia is rare in pregnancy, occurring in up to 1 in 100,000 pregnancies. A 28F G2P1 was diagnosed at our institution with Philadelphia (Ph) negative B-ALL at 24 weeks' gestation. She was induced with UKALL14 and achieved a flow negative complete remission post-induction. During induction chemotherapy, regular ultrasound scans showed that the fetus was growing normally for gestational age. The patient was kept on weekly vincristine and prednisone and had an elective c-section at 31+3 weeks, in light of her previous caesarean section, poor bishop score, and in order for her to safely resume chemotherapy. Baby girl had an uneventful hospital course and was discharged after 29 days. Given the sparse data on this challenging topic, we decided to review the literature for all reported cases of ALL in pregnancy. Methods : We reviewed the literature for all reported cases of ALL from 2000 to 2023. We collected available data on the patient, pregnancy, and fetal outcome as well as leukemia characteristics and chemotherapy received. Results: We found 57 patients across 20 studies. The median age at diagnosis was 26 years (range 16-41 years). Most affected pregnancies were during the second trimester (T2) (n 23/57) and third trimester (T3) (n 20/57). The subtype of ALL was reported in 49 patients; of those, B ALL was the most common (n 35/49). Ph was reported positive in eight patients. A total of 53 patients received chemotherapy, of those 33 patients were received during pregnancy, while 13 were after pregnancy, and the remaining 7 had unclear timing. Patients received different combinations of standard ALL chemotherapy agents including vincristine (n 49/53), cyclophosphamide (n 25/53), prednisone (n 42/53), dexamethasone (n 23/53), L-asparaginase (n 20/53), pegaspargase (n 1/53), daunorubicin (n 36/53), doxorubicin (n 7/53), idarubicin (n 4/53), cytarabine (n 11/53), 6-mercaptopurine (n 8/53), cytosine arabinoside (n 1/53), and methotrexate was given to 8 patients intrathecally during pregnancy; 6 during T2 and 2 during the first trimester (T1). Dasatinib and imatinib were given to two patients at 33 and 24 weeks respectively. There were no reports of patients who were treated with immune therapy while pregnant. All four patients who did not receive any treatment died from progressive disease. Complete remission (CR) was reported in 45 patients. One patient died during induction from septicaemia, five were refractory to induction chemotherapy and 2 did not have induction outcome reported. Of those who achieved a CR, 21 were reported alive and 16 reported dead at last follow up. Among those mortalities, 8 were due to relapsed leukemia, 3 due to infectious causes, 2 due to respiratory failure and 2 from unspecified causes. Five patients were reported to have persistent or refractory leukemia, 4 of whom died. Among the 14 T1 pregnancies, 10 were terminated electively, 3 spontaneously, and there was one maternal death due to declining treatment. Three patients received chemotherapy during T1, two suffered a spontaneous abortion and one had a missed miscarriage. Among the 23 T2 pregnancies, 17 resulted in live deliveries, one stillbirth during chemotherapy, one maternal death and 4 spontaneous abortions; 2 of which received chemotherapy during pregnancy, one did not receive treatment and one abortion with unclear timing of chemotherapy initiation. Among the 20 T3 pregnancies, 19 had successful deliveries and one stillbirth at an unindicated gestational age after receiving chemotherapy. Five babies were reported to have low birth weight, four had neonatal cerebral ischemia, one had intraventricular hemorrhage and one suffered from intestinal obstruction 2 weeks after delivery. All babies were discharged from hospital with no adverse congenital or developmental complications. Conclusion : Treatment of women presenting with ALL at or beyond T2 is possible with high early remission rates for the mothers and delivery of healthy babies. The outcome is highly dependent on the gestational age. It is important that more cases are reported on this topic due to its challenging and multidisciplinary nature especially given the sparse data available and potential for reporting bias.

Increasing Palliative Care Team Involvement in Pediatric Hematopoietic Stem Cell Transplant Patients

Background/Objectives: Palliative care facilitates communication, helps with physical and psychological symptom management, and assists in goals of care and advance care planning discussions. Multiple organizations including the World Health Organization, American Academy of Pediatrics and the American Society of Clinical Oncology encourage palliative care involvement. Palliative care involvement with hematopoietic stem cell transplant (HSCT) patients has been shown to be beneficial as HSCT is associated with a high degree of morbidity and possible mortality. We sought to increase the frequency of palliative care consultations for patients planning to undergo HSCT for targeted diagnoses. The outcome measure was percentage of patients who underwent HSCT for a targeted diagnosis who had a Palliative Care Team (PaCT) consult. Baseline data revealed 48 patients underwent HSCT from July 2020 to July 2022, 25 of whom had a targeted diagnosis. PaCT met 12 of the 25 patients (48%). Our goal is to increase the number of PaCT consults for patients receiving HSCT for our targeted diagnoses from 48% to 75% by December 2023. Methods: Chart review was conducted for patients who underwent HSCT from July 2020 to July 2022. Demographics documented included reason for HSCT, if PaCT was consulted, date of transplant, date of PaCT consult and living status. A targeted diagnosis list was compiled based on which patients were felt to benefit most from palliative care involvement: relapsed or refractory leukemias and lymphomas, myelodysplastic syndrome, high risk acute myeloid leukemia and metabolic disorders, such as Hurler syndrome. The first plan-do-study-act (PDSA) cycle included discussion of the project at division meetings, posting flyers in the clinic provider workrooms, and education regarding palliative care and evidence of its involvement in HSCT patients. The second PDSA cycle implemented involved revision of the HSCT referral form to include the question “Is PaCT consulted.” If the provider answers “no,” further elaboration is requested. Results: From 1/1/23 to 7/31/23 ten patients with a targeted diagnosis underwent HSCT. Five patients (50%) received a PaCT consult prior to HSCT. Conclusions: Palliative care involvement in pediatric oncology is well established, but its role in HSCT patients continues to be explored. As HSCT patients receive more intense therapy, have frequent deaths in intensive care units and can have limited opportunity for end of life planning due to rapidly changing clinical courses, early integration of PaCT could allow for decreased symptom burden and distress for both patients and families. Future PDSA cycle interventions include adding PaCT consultation to the transplant evaluation order set in the electronic medical record and review of the comments on the completed HSCT referral forms to identify possible barriers in the PaCT consultation process.

Transcriptomic without Clinical Response to Menin Inhibition As a Mechanism of Upfront Resistance in Samples from Mutliply Relapsed Patients with KMT2A-Rearranged Leukemia

Inhibitors of the Menin-KMT2A interaction are emerging as promising agents for the treatment of KMT2A-rearranged ( KMT2A-r) acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Menin is required for the recruitment of the KMT2A-fusion to a subset of its target genes. Early phase clinical trials have shown highly encouraging results in patients with relapsed and refractory leukemias with KMT2A rearrangements and NPM1 mutations. In a recently reported early phase clinical trial evaluating the Menin inhibitor revumenib, about 50% of patients failed to show a clinical response despite robust downregulation of known Menin:KMT2A target genes, suggesting on target activity of the drug. This was the most common pattern of upfront resistance. We sought to understand upfront resistance (in contrast to the reported induced resistance via MEN1 mutations) in patient derived xenograft models. To this end, we evaluated the efficacy of Menin-inhibition in KMT2A-r leukemias with high-risk features: high-risk KMT2A fusion partners in AML ( MLLT10, MLLT4), infant ALL, and serial samples from patients with multiply relapsed ALL. We found that AML samples with high-risk fusion partners banked at initial diagnosis were sensitive to Menin-inhibition in xenografts. In contrast, three highly pretreated samples from patients with KMT2A-AFF1 ALL showed only a slight, or no decrease in latentcy in the Menin-inhibitor arm. Interestingly, Menin inhibition showed excellent in vivo efficacy against leukemia samples obtained from the same three patients earlier in their disease course. Therefore, prior chemotherapy (+/- immunotherapy) selects for Menin-inhibitor resistant cells without prior exposure to Menin inhibitor. Transcriptomic analysis (RNA-Seq) documents sustained on-target efficacy of the Menin inhibitor on a transcriptional level in resistant cells. None of the samples contained a MEN1 mutation at any time point. Targeted panel sequencing at initial diagnosis and relapse documented clonal evolution and the emergence of multiple co-mutations (particularly RAS pathway mutations and TP53 mutations), however, all co-mutations tested failed to induce resistance as a single alteration. It is possible that the cooperation of multiple emerging mutations (rather than a single mutation) is sufficient to drive AML in a KMT2A-fusion independent manner, thus mediating Menin resistance. A transcriptomic response (i.e. downregulation of KMT2A-fusion target genes) without a clinical response was the most common pattern of upfront resistance in the recently reported phase I/II clinical trial of the Menin inhibitor revumenib. We conclude that it will be critical to use Menin-inhibitors upfront or in early lines of therapy before substantial clonal evolution has occurred.

Kdm6a Modulates Hematopoiesis and Leukemogenesis Via Demethylase-Dependent Epigenetic Programming

Hematopoietic stem and progenitor cells (HSPCs) maintain their differentiation by precisely regulating gene expression patterns at the transcriptional and epigenetic levels. KDM6A encodes an epigenetic regulator of the repressive chromatin mark histone 3 lysine 27 tri-methylation (H3K27me3). Growing evidence suggested the involvement of KDM6A in hematological malignancies. KDM6A mutations in leukemia frequently occur in males, reflecting that KDM6A escapes X chromosome inactivation. Therefore, female patients with single-allele loss-of-function KDM6A mutations will partially retain KDM6A expression, which is called haploinsufficiency. Furthermore, KDM6A haploinsufficiency was reported to be associated with refractory and/or relapsed leukemia, while the role of KDM6A haploinsufficiency in hematopoiesis remains unexplored. We established kdm6a-null zebrafish using CRISPR-Cas9 gene editing. We found that kdm6a loss impaired HSPCs formation. kdm6a-null embryos showed reduced expression of the hemogenic endothelial and HSPC markers runx1 at 28 hours post-fertilization (hpf). Using Tg(kdrl:mCherry;cmyb:en-GFP) embryos, we also found that the number of hemogenic endothelium and emerging HSPCs in kdm6a-null was significantly decreased. To study the importance of Kdm6a-mediated demethylase activity, we ectopically expressed kdm6a constructs (wild-type kdm6a, or catalytically-dead kdm6a) in kdm6a-null embryos. The result showed that, unlike restoring effect by wild-type kdm6a, the ectopic expression of catalytically-dead kdm6a constructs ( kdm6a H1134A) showed no rescue effect for decreased HSPC numbers. Then, we aimed to understand the potential role of kdm6a haploinsufficiency in hematopoiesis and leukemogenic mechanisms. We found that zebrafish mutants that are haploinsufficient in kdm6a ( kdm6a +/-) exhibited uncontrolled myeloid differentiation. The expression of myeloid-specific markers substantially increased in kdm6a +/- mutants, compared with WT siblings at 72 hpf. Confocal analysis showed that the ratio of myeloid-primed HSPCs was markedly increased in kdm6a +/- embryos compared to WT siblings. Consistently, a significant upregulation in transcripts encoding key myeloerythorid TFs including cebps, irf8 and gata1a was observed in kdm6a +/- embryonic HSPCs. Furthermore, kdm6a +/- adults exhibited the phenotype of monocytosis, which mimics chronic myelomonocytic leukemia, or exhibited the phenotype of increased progenitors, which mimics acute myeloid leukemia. We next sorted HSPCs from WT or kdm6a +/- at 72 hpf for RNA-sequencing and ATAC-sequencing. Enrichment analysis showed transcriptional dysregulation of genes involving “Purine metabolism”, “Cysteine and methionine metabolism”, “TGF-beta signaling pathway”, and “Cell cycle”. GSEA further uncovered positive enrichment of gene sets in kdm6a +/- embryonic HSPCs, including “Oxidative phosphorylation”, “mTORC1 signaling” and “DNA repair”. These dysregulated pathways in kdm6a +/- HSPCs are highly involved in cellular homeostasis and hallmarks of aged hematopoietic system. ATAC analysis also revealed robust changes in chromatin accessibility, especially including genes related to aged hematopoietic system. Integrated RNA-seq and ATAC-seq analyses showed that the chromatin accessibility at promoters and transcription of 63 genes were decreased in kdm6a +/- HSPCs. And we found that the negative regulator of Jak-Stat signaling- socs3a was on the list. Ectopic expression of socs3a in kdm6a +/- could partially rescue the uncontrolled myeloid differentiation of HSPCs. Consistently, abnormal myeloid differentiation from kdm6a +/- mutations was rescued by Jak or Stat3 inhibitors. We also tested the importance of Kdm6a-mediated demethylase activity. Unlike restoring effect by wild-type kdm6a, the ectopic expression of catalytically-dead kdm6a constructs ( kdm6a H1134A) showed no rescue effect for dysregulated expression of socs3a and key myeloerythorid genes including cebps. In conclusion, our data demonstrate the multiple roles of kdm6a in regulating HSPC homeostasis and provide new insight into the mechanism of kdm6a haploinsufficiency in uncontrolled myeloid differentiation through demethylase-dependent epigenetic programming.

Multinational Retrospective Analysis of Bridging Therapy Prior to Chimeric Antigen Receptor T Cells for Relapsed/Refractory Acute Lymphoblastic Leukemia in Children and Young Adults

Background: Chimeric antigen receptor (CAR) T cells targeting CD19 are a well-established treatment option for children and young adults suffering from relapsed and/or refractory B-lineage acute lymphoblastic leukemia. Nonetheless, there is still insufficient data about the proper management of bridging therapy between eligibility for therapy and administration of CAR T cells taking into consideration that most of the patients are heavily pretreated. Bridging therapy has been designed to achieve a low leukemia burden prior to CAR T cell infusion. However, systematic data of bridging therapy are still limited and the effect of bridging therapy on outcome, side effects and response to CAR T cell therapy is still poorly understood. With this retrospective, multinational, large-scale study, we strive to understand the impact of low- and high-intensity bridging regimens on a variety of outcome parameters in order to improve the basis for clinical decision making in bridging therapy prior to CAR T cell administration. Methods: Real-world data were collected from 83 patients receiving 88 CAR T cell therapies from twelve different sites in Germany, Austria and Switzerland. Data were collected anonymously via paper case report forms and subsequently analyzed. Performed treatments were classified into the categories 1) no systemic therapy, 2) low-intensity therapy and 3) high-intensity therapy. Bridging therapies were defined as high-intensity if at least one chemotherapeutic agent of the following was given: cyclophosphamide/ifosfamide, etoposide, anthracyclines or other agents with high toxicity potential (intravenous methotrexate, platinum-based antineoplastic drugs, thiotepa, high-dose cytarabine, fludarabine). Low-intensity bridging therapies comprised the administration of steroids, vincristine, low-dose cytarabine, PEG-asparaginase/Erwinia asparaginase and oral maintenance therapy (mercaptopurine, thioguanine, oral methotrexate, hydroxyurea). The administration of specific chemotherapeutic agents as well as immunotherapies and targeted therapies was assessed. CAR therapies comprised CD19 2 nd generation CAR T cell products from commercial and academic providers. We then analyzed the impact of different bridging regimens on several outcome parameters such as overall and disease-free survival, adverse events, tumor burden and performance status at defined time points (eligibility, leukapheresis if performed, lymphodepletion/CAR T cell infusion). Results: 33 of 88 treatments were classified as high-intensity and 34 as low-intensity bridging regimens. Prior to 13 CAR T cell administrations no systemic bridging therapy was given, 8 of 88 bridging regimens could not be stratified due to incomplete data (Figure 1). Between eligibility and apheresis, mostly low-intensity therapy or no systemic therapy was given. Within the period between apheresis and CAR T cell infusion, treatment diversified due to the heterogeneity of the cohort. Patient characteristics are listed in Table 1. Patients receiving a high-intensity bridging therapy had a significantly higher tumor burden at time point of eligibility defined by blasts in bone marrow and by measurement of minimal residual disease (MRD) compared to patients treated with a low-intensity or no systemic bridging therapy. Tumor burden within the two groups converged over the time of bridging therapy. However, at time of lymphodepletion, patients in the high-intensity group showed a significantly lower performance status indicated by Karnofsky/Lansky score than patients in the low-intensity/no systemic therapy group, reflecting the higher toxicity potential. Furthermore, these patients suffered significantly more often from bacterial adverse events and mucositis. Neither overall nor disease-free survival differed significantly between the two bridging regimen groups. Conclusion: In this retrospective cohort data, a high-intensity bridging therapy has not improved the outcome of CAR T cell therapy in terms of overall and disease-free survival. Yet high-intensity bridging therapy has caused more mucositis, bacterial adverse events and worsened the performance status. Our study suggests that a low-intensity bridging regimen may be preferred whenever tumor burden and disease kinetics allow this treatment strategy.

Molecular Attributes of CAR T Cell Fitness in Patients with Chronic Lymphocytic Leukemia

Chimeric antigen receptor (CAR) T cell therapies have transformed the treatment of hematologic malignancies, leading to FDA approval in B-cell acute lymphoblastic leukemia, non-Hodgkin lymphoma, and multiple myeloma. While many patients with previously untreatable disease have achieved excellent responses and long-term remissions, a substantial number do not respond or relapse after months to years. Most prior studies of CAR T cell-intrinsic determinants of response and resistance have focused on infusion products. Functional expansion and persistence of CAR T cells in patients constitute an important feature of treatment success, yet the trajectories of CAR T cells and their molecular underpinnings remain poorly understood due to technical challenges. Here we performed single-cell multiomics with T-cell receptor (TCR) lineage tracing to study the molecular attributes of CAR T cell fitness, which we define as the ability to expand and/or persist to at least 12 months after infusion. We recently published long-term follow-up from a prospective single-center phase 2 clinical trial in which CD19-directed CAR T cells with 4-1BB co-stimulation were administered to patients with relapsed or refractory chronic lymphocytic leukemia (CLL) not in complete remission despite at least 6 months of ibrutinib. In this trial, we found high rates of deep and durable remissions with prolonged persistence of functional CAR T cells determined by flow cytometric detection of CAR T cells with concomitant B-cell aplasia. We obtained CAR T cell infusion products and peripheral blood at day 10 and month 12 from five patients with durable remissions. We then performed single-cell multiomics sequencing to measure gene expression, TCRs, surface proteins, and chromatin accessibility to define the multidimensional phenotypes and clonal trajectories of CAR T cells over time ( Figure). We first evaluated CAR T cell dynamics from infusion product to day 10 and identified clones that expanded using TCR as a molecular barcode. While ~90% of CAR T cells in the infusion product are CD4 +, peak expansion at day 10 is driven by effector CD8 + CAR T cells. Clonally expanded CAR T cells at day 10, compared to non-expanded CAR T cells, showed higher expression of multiple cytotoxicity genes ( GZMH, GZMB, GNLY, PRF1) and co-inhibitory receptors ( LAG3, PDCD1, and KLRB1) and lower expression of memory genes ( SELL, LTB, TCF7). Using TCR lineage tracing and a machine learning classifier, we identified similar markers predictive of clonal expansion from the infusion product, e.g. CD8A, GNLY, KLRG1, KLRK1 among the top positive markers and CD4 and SELL among the top negative markers. Along with clonal expansion, we found that CD8 + CAR T cells undergo significant evolution in their chromatin accessibility landscape from infusion product to day 10. Genomic regions that are more open at infusion are enriched for Fos/Jun (AP-1) and Batf family of transcription factor (TF) motifs, consistent with their function in promoting effector T cell differentiation and proliferation. Genomic regions that are more open at day 10 are enriched for Eomes, T-bet, and other T-box TF motifs, consistent with their roles in regulating T cell cytolytic function, memory formation, and exhaustion. These data provide insights into the regulatory mechanisms of rapid CAR T cell expansion post-infusion in patients which have not yet been reported. We found that most clonally expanded CAR T cells at day 10 contracted or disappeared by month 12. However, a subset of CD8 + CAR T cells in 2 patients that did not undergo early expansion surprisingly demonstrated late clonal expansion and persistence at month 12, coinciding with hyper-expansion (>200 cells per clone) of their TCR clonotypes with or without CAR expression. We then investigated the specificity of TCRs using publicly available databases and found late expansions of clones predicted to bind viral peptides. This observation suggests that the TCR antigen specificity may contribute to the long-term persistence or fitness of CAR T cell clones, in contrast to recent studies of long-lived CAR T cells in a limited number of patients which argue that persistence is driven by specific phenotypes rather than clonotype-specific features. Further validation of this finding is ongoing through flow enrichment of rare persistent CAR T cells for deeper single-cell sequencing and in vitro cellular immunology assays.

Intrathecal Chemotherapy As Treatment for Chimeric Antigen Receptor T Cell (CAR T) Therapy Associated Neurotoxicity

Chimeric antigen receptor T cell (CAR T) therapy is offering high remission rates for patients with relapsed and refractory lymphomas, myelomas and acute lymphoblastic leukemias. CAR T therapy causes significant morbidity and mortality associated with its common side effects of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Steroids remain the standard first line treatment for ICANS, however many patients either do not respond or develop rebound symptoms as steroids are tapered. Moreover, patients with severe ICANS who require higher cumulative doses of steroids tend to have worse progression-free survival and overall survival. To mitigate the toxicity associated with steroids use, we have increasingly added intrathecal chemotherapy as part of the treatment algorithm for patients with ICANS who are not responsive to steroids or with grade3-4 ICANS at diagnosis. we hereby report on the outcomes of 12 patients who received intrathecal chemotherapy for the treatment of ICANS at our center between 2017 and 2023. A total of seventy six patients with Non-Hodgkin's lymphoma (Follicular n=6, Primary mediastinal n=1, DLBCL n=69) received CAR T therapy at our center with a median age of 63.5 years (31, 86). Median follow up for survivors was 16.9 ( 3.3, 65.9 months)Patients characteristics were male 62%, race ( white 83%, black 12%), primary induction failure (44%), received axicabtage ciloleucel (n=24,32% ), lisocabtage (n=28, 36%) and tisagenlecluecel (n=24, 32%). Forty-four patients (59%) developed CRS (grade 1/2 in 89% of CRS cases) with a median time to CRS of 3 days (0,15) and a median duration of 3 days (0,36). Thirty-three patients (43%) developed ICANS with a median onset of 5 (0,19) days and median duration of 3.5 (0,28) days. The maximum ICANS grade was grade 1 (n=14, 43%), grade 2 (n=6, 18%), grade 3 (n=11, 33%) and grade 4(n=2, 6%). Twelve patients received intrathecal chemotherapy with 10/12 receiving one dose and 2 patients requiring 2 dosages. Patients who received intrathecal chemotherapy had a median age of 65(47,79) years, male (n=8,67%), Median time from diagnosis to IEC 340(214, 5374 days), and ICANS (grade 1 n=4, grade 2 n=2 and grade 3 n=6). Median time to ICANS was 5 (2,13) days and median ICANS duration was 4(1,25) days. Methotrexate was used in 10 patients and cytarabine in 2 patients. All patients received steroids and 6 patients (50%) received Anakinra as part of ICANS management. Median time for ICANS to receiving intrathecal chemo was 1 (1,25) days with 8 patients receiving intrathecal in the first 24 hours and 2 patients within 48 hours within developing ICANS. Eleven patients had resolution of their ICANS with median time to resolution of 2 days (1,24). Five patients had complete resolution within 24 hours of intrathecal chemotherapy. Five patients had no response to steroids, and all had resolution of their ICANS symptoms after intrathecal chemotherapy. Our data shows that early administration of intrathecal chemotherapy is feasible and is highly effective in management of ICANS. These findings will need further validation in larger trials to reduce ICANS toxicity and the added adverse events of systemic steroids and immunosuppression.

Impact of Salvage and Bridging Therapy in Adult Patients with Refractory B-Cell Acute Lymphoblastic Leukemia (B-ALL) Referred for Anti-CD19 CAR T-Cell Therapy: An Intention to Treat Analysis

Background: Prognosis of relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (ALL) is poor, particularly after allogeneic hematopoietic cell transplantation (alloHCT). In this setting, several CAR T-cell products targeting CD19 (CART19) have been able to achieve complete response (CR) rates of 70 to 90%. However, a significant proportion of these patients still relapse. Several studies focus on the effect of the CART19 product on efficacy, however, information on how salvage therapy (ST) and bridging therapy (BT) can modulate the outcomes of CART19 therapy is scarce. Methods: We report the outcomes of all adult patients with R/R B-ALL intended for treatment (aphaeresis performed) with available CART19 products in a single institution from Jul 2017 to Jul 2023. This includes patients intended to receive the academic varnimcabtagene autoleucel (var-cel) in the CART19-BE-01 trial and the consecutive compassionate use and hospital exemption programs, as well as with commercially available tisagenleuceucel (tisa-cel). Lymphodepletion was performed with fludarabine (90 or 120 mg/m 2) and cyclophosphamide (900 or 1000 mg/m 2), followed by the infusion var-cel or tisa-cel, respectively. We analyzed all ST (treatment administered after last relapse before CART19-apheresis) and BT (treatment administered after apheresis and before CART19 infusion) performed, and analyzed the impact of BT on PFS and OS after apheresis. Results: A total of 74 adult B-ALL patients underwent leukapheresis and CART19 (var-cel/tisa-cel) production. At screening patients had a median age of 34 years (19-78), 45% were females, with a median of 3 prior lines of therapy (2-7) including inotuzumab (69%), blinatumomab (27%) and allo-HCT (84%). After last relapse, 92% of patients received ST with a median of 1 (0-3) treatment line, including inotuzumab (48%), high-dose chemotherapy (28%), low-dose chemotherapy (21%), TKIs (12%), blinatumomab (9%), radiotherapy (7%) and DLIs (4%). The overall response to ST prior apheresis was MRD-negative CR (28%), MRD-positive CR (22%), partial response (4%) and progressive disease (PD) in 46%. After apheresis was secured, BT was performed in 76% of patients with a median of 1 (0-2) bridging line, including low-dose chemotherapy (44%), intrathecal therapy (23%), high-dose chemotherapy (15%), TKIs (15%), inotuzumab (14%), radiotherapy (3.5%), blinatumomab (1.8%) and DLIs (1.8%). BT was not administered in 24% (8/74) of patients mainly due to morphological remission at screening (83%) and frailty (11%). The overall response to BT prior CART19 infusion was MRD-negative CR (31%), MRD-positive CR (12%), partial response (7%) and progressive disease (47%), with a 3% treatment related mortality (TRM) events attributed to BT. Inotuzumab administration occurred either as ST or BT to CART19 in 86% of patients (the remaining 14% was previouly given as bridge to allo-HCT), and was able to achieve MRD-negative CR in 42% of patients. The overall control of the disease after BT either worsened (51%), remained similar (14%), or improved (35%). After this, 12% (n=9) of CART19 treatments were not performed due to progressive disease (33%), TRM (22%), therapeutic alternative (22%), and production failure (11%). Infused patients achieved an MRD-negative CRR of 90% with a median PFS of 11,2 months. There were no differences in OS between patients with ≥5% or <5% bone marrow (BM) blast counts either before apheresis (ITT, figure 1) or after BT prior to CART19 infusion. However, despite the lack of apparent impact of this disease burden categorization on the OS at both timepoints, a statistically significant difference was observed after categorizing patients regarding the response to BT (figure 2), with a median PFS of 6.6 vs 18.6 months if the overall disease status of the patients either worsened or remained stable/improved after BT (p=0.0002). Conclusions: To the best of our knowledge this could be one of the first studies focusing on the impact of ST and BT in adult patients with R/R B-ALL referred for CART19 therapy. These results suggest that the potentially synergistic impact of bridging therapy may be mediated more by the modulation exerted by BT on the proliferative kinetics of the disease rather than on the absolute tumour burden of the patient. More detailed results on the independent impact of each type of BT will be provided in the final presentation.

CD22 CAR T Cell-Related IEC-HS Is Associated with an IFN-γ Cytokine Signature

Introduction Chimeric antigen receptor T-cell therapy targeting CD22 (CAR22) shows promise for patients with relapsed / refractory B-cell acute lymphoblastic leukemia (ALL) and large B cell lymphoma (LBCL) (Fry et al. Nat Med 2018; Baird et al. Blood 2020). The overall response rate for CAR22 in adults who received prior CD19 CAR therapy is 100% and 68% in ALL and LBCL respectively (Tandem 2023) (NCT04088890). However, 33% of pediatric ALL patients who received CAR22 developed immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS) (Shah et al. JCO 2020). IEC-HS is characterized by hyperferritinemia, cytopenias, coagulopathy and end organ damage. Pediatric ALL data suggest many cytokines including IFN-γ, IL-1β, IL-18 and TNF-α are upregulated in IEC-HS (Ishii et al. JCI 2020, Lichtenstein et al. Blood 2021). Here, we report the clinical features and cytokines associated with IEC-HS after CAR22 infusion in predominantly adult subjects with ALL and LBCL. Methods Plasma was collected weekly from Day 0 to 28 for 54 patients (ALL: n = 16, LBCL: n = 38; adult: n = 51, pediatrics: n = 3) who were treated with CAR22 manufactured using an automated, closed-system Miltenyi CliniMACS Prodigy device. Three dose levels were tested (ALL: 3 x 10 5 cells/kg, LBCL dose level 1 (DL1): 1 x 10 6 cells/kg, LBCL dose level 2 (DL2): 3 x 10 6 cells/kg). Clinical characteristics associated with IEC-HS were retrospectively collected and graded according to consensus criteria (Hines et al. TCT 2023). Eighty cytokines were quantified in batch via multiplexed fluorescence immunoassay (Luminex) to generate mean fluorescent indices (MFIs). Unsupervised hierarchical clustering using Euclidean distance and ward.D2 linkage were employed to analyze cytokine dynamics. Statistical analyses were performed using R (v 4.2.2). Results 8 of 54 (15%) patients developed IEC-HS, all were ≥18 years old. 5 patients had LBCL, 3 had ALL. 5 cases were grade 2, 3 were grade 4. Median onset was D+12 (range 8 - 21). All patients had prior CRS with median onset at D+1 (range 0 - 9). The 3 ALL patients had ongoing CRS and spectral overlap with IEC-HS at time of diagnosis whereas LBCL patients' CRS resolved prior to IEC-HS onset. IEC-HS patients had significant increases in inflammatory markers (ferritin, CRP, LDH, triglycerides), coagulation markers (INR, PTT), cytopenias (hemoglobin, platelets) and transaminases (Table 1). Patients most commonly received high-dose glucocorticoids, tocilizumab and anakinra. Additional agents for grade 4 IEC-HS included anti-thymoglobulin, dasatanib and emapalumab. 5 of 8 patients developed serious infections requiring IV antimicrobials including bacteremia, invasive fungal infections and viral reactivation. 5 patients with IEC-HS died after achieving complete response: 3 due to IEC-HS and concomitant infection, 1 from t-AML and 1 lost to follow up. IEC-HS occurred more frequently at higher doses in LBCL where multiple doses were tested (DL2: 33% vs DL1: 7%, p = 0.08). Peak serum CAR22 concentration was markedly elevated in patients who developed IEC-HS compared to those who did not (median 648 vs 42 cells/µL, p<0.001). Unsupervised hierarchical clustering performed on serial cytokine measurements identified a signature of IFN-γ-related cytokines including CXCL9, CXCL10, TNFα, FasL, IL6 and IL18 as being closely related in IEC-HS (Figure 1). Most cytokines in the signature peaked at D+14, consistent with onset of clinical IEC-HS. Peak levels of nearly all cytokines in the interferon-γ cluster were significantly elevated in IEC-HS patients compared to patients who did not develop IEC-HS, and this cluster contained most cytokines with significant increases in peak levels (Figure 1). Conclusions IEC-HS is a potentially life-threatening toxicity associated with CAR22 across age and underlying malignancy. The high rates of non-relapse mortality and severe infections in this study highlight the importance of understanding the immune dysregulation underlying IEC-HS. Higher CAR22 dose and expansion were associated with IEC-HS, indicating optimal dosing as a potential preventive strategy. The observed IFN-γ cytokine signature is consistent with myeloid cell activation and Th1 T-cell skewing that likely contributes to IEC-HS pathogenesis. These cytokines may be amenable to therapeutic intervention and further study of approaches targeting JAK/STAT and IFN-γ are warranted.

Covalent Menin Inhibitor Bmf-219 in Patients with Relapsed or Refractory (R/R) Acute Leukemia (AL): Preliminary Phase 1 Data from the Covalent-101 Study

Background: Menin, a protein involved in transcriptional regulation, plays a role in the genesis of multiple cancers. Preclinical data from BMF-219, an investigational, highly selective, oral small-molecule inhibitor of menin, show sustained potent abrogation of menin-dependent oncogenic signaling. BMF-219 is the first and only covalent menin inhibitor in clinical development and is being evaluated in multiple hematologic malignancies, solid tumors, and diabetes mellitus. COVALENT-101 (NCT05153330) is a Phase I dose-escalation and -expansion study of BMF-219 in R/R AL (Cohort 1), DLBCL (Cohort 2), MM (Cohort 3), and CLL (Cohort 4). Here we report preliminary safety, PK and anticancer activity data from Cohort 1 (AL). Methods: Doses of BMF-219 are escalated independently for each indication, initially in single-subject cohorts followed by a “3 + 3” design. Eligible patients (pts) include adults with R/R AL ineligible for standard therapy. Initially pts were enrolled agnostic to molecular status. A subsequent amendment introduced quotas for KMT2Ar (MLL1r), NPM1 and other known menin-dependent mutations: CEBP/A, MLL1-PTD, MN1, NUP98, NUP214, PICALM-AF10, SETBP1. Prior exposure to reversible menin inhibitor therapy is permitted. Subjects receive BMF-219 daily for continuous 28-day cycles until progression/intolerability. There are 2 parallel dose-escalation arms: pts not taking (Arm A) or taking (Arm B) moderate or strong CYP3A4 inhibitors. The study is ongoing and accruing in the escalation. Expansion cohorts will enroll pts to obtain further safety and efficacy data at the OBD/RP2D. Results: As of data cutoff of 7/24/2023, 26 pts with R/R AL (24 AML; 2 ALL) are enrolled; 7 remain on study treatment. Baseline characteristics include 17(65%) males and 9(35%) females with a median age of 57.5 years (range 33-84). There is a median of 4 (range 1-8) prior lines of therapy and 11 (42%) with prior HSCT(s). Six pts (23%) had KMT2Ar, 3 (12%) KMT2A-PTD, 4 (15%) NPM1, and 13 (50%) WT for KMT2A and NPM1. Dosing began with single-patient cohorts at 100 mg QD (Arm A) and 25 mg QD (Arm B) and has been escalated through 4 dose levels. Thus far, pts have been dosed up to 500 mg QD (Arm A) and 125 mg QD (Arm B). BMF-219 exposures were comparable between arms, with ~2-4-fold higher exposures observed with co-administration of a moderate or strong CYP3A4 inhibitor. At the highest dose (DL4) in which PK was evaluated, Arm A (500 mg QD) and Arm B (125 mg QD), pts on average achieved ~50% of target exposure (2000 ng*hr/mL) with some pts surpassing it. Higher QD dosing or corresponding BID dosing is expected to achieve desired exposure. BMF-219 has generally been well tolerated with no DLTs observed and no discontinuations due to treatment-related toxicities. No related QTc prolongation was observed. At the time of data cutoff, 23 of 26 pts were included in the safety population. Common TRAEs (≥10%) include vomiting 13% (3) and Differentiation Syndrome (DS) 13% (3). No Grade 5 TRAEs were reported. The only common Grade ≥3 TRAE (≥5%) was DS 13% (3). The efficacy evaluable population includes AML pts who meet the following criteria: dosed at or near predicted efficacious dose (500 mg or above [Arm A]; 125 mg or above [Arm B]), had known menin-dependent mutations, and completed at least one scheduled response assessment (or had a minimum of 7 doses if discontinued prematurely). Thus far, 2 of 5 efficacy evaluable patients achieved a complete remission (1 CR; 1 CRi) and both continue BMF-219 treatment. Patient A: 39/M, NUP98-NSD1, ECOG=0, 500 mg QD, Arm A, 4 prior lines of treatment including intensive chemotherapy and allo-HSCT. At C1D27, marrow blasts were reduced to 6% from 13% at study entry. The patient achieved CR at C2D28 with 0% blasts.Patient B: 70/F, NPM1m, ECOG=1, 125 mg QD, Arm B, 1 prior line of treatment with decitabine and an investigational agent. At C1D28, marrow blasts were reduced to 34% from 52% at study entry. The patient achieved CRi with 3% blasts at C2D28. Conclusion: BMF-219 is generally well tolerated with no DLT observed (and able to be taken with and without CYP3A4 inhibitors) with no pts discontinuing therapy due to toxicity. BMF-219 dose escalation is ongoing and approaching target exposure. BMF-219 demonstrates early signs of clinical activity in different genomic subgroups. The trial is ongoing and includes enrollment for pts diagnosed with AL, DLBCL, MM and CLL.

Toxicity, Progression-Free Survival, and Quality of Life of Patients Treated with Zanubrutinib Versus Ibrutinib: A Q-TWiST Analysis from the ALPINE Study in Relapsed or Refractory Chronic Lymphocytic Leukemia

Introduction: Zanubrutinib is a second-generation Bruton's tyrosine kinase inhibitor (BTKi) with enhanced specificity over the first-generation BTKi ibrutinib. Patients treated with zanubrutinib had a significantly longer progression-free survival (PFS) versus those treated with ibrutinib in the global, phase 3, randomized ALPINE trial in patients with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) (NCT03734016). Quality-adjusted time without symptoms of disease and toxicity (Q-TWiST) is a clinical tool to assess overall benefits and risks of cancer therapies by integrating progression, survival, treatment toxicity, and patient quality of life (QoL) into a single metric. It provides valuable insights into the quality and duration of improved health states. To gain a comprehensive understanding of the benefits and risks associated with zanubrutinib versus ibrutinib in terms of quality-adjusted survival, a Q-TWiST analysis was performed within the ALPINE trial. Methods: Patients in the ALPINE trial were followed for a median duration of 29.6 months. For the Q-TWiST analysis, overall survival (OS) of each patient was partitioned into 3 mutually exclusive health states: TOX (time before disease progression with toxicity after randomization); TWiST (time from randomization to disease progression without toxicity); and REL (time after disease progression until death or censoring). Survival curves corresponding to TOX, PFS, and OS were estimated by Kaplan-Meier method with a monthly cycle. Restricted mean survival time for each health state was derived from the area under the Kaplan-Meier curve (TWiST = PFS - TOX; REL = OS - PFS). Q-TWiST for each treatment was estimated as the mean time spent in each health state weighted by its respective QoL, denoted by health states' utility value (U; 0 [indicates death] to 1.0 [indicates “perfect” health]). Relative Q-TWiST gain was estimated by dividing the absolute Q-TWiST gain by mean OS with ibrutinib. To ensure the comparability of populations and treatments across published Q-TWiST studies, the base-case analysis focused on a high-risk population (i.e., patients with chromosome 17p deletion, TP53 mutation, or both). In the base case, the TOX health state included adverse events (AEs) of grade 2 or higher. Standard utilities were applied, with a value of 0.5 assigned to both TOX and REL and a value of 1.0 assigned to TWiST. A sensitivity analysis was performed in an intent-to-treat population with same-grade AEs considered for TOX and standard utilities. Results:In the base case, the mean durations of TOX, TWiST, and REL health states for zanubrutinib (N = 73) were 11.54 months, 14.45 months, and 1.70 months, respectively. The corresponding durations for ibrutinib (N = 71) were 11.38 months, 11.09 months, and 3.78 months, respectively. The mean difference (95% CI) for zanubrutinib versus ibrutinib was 0.16 months (−0.18, 0.51) for the TOX state, 3.36 months (2.6, 4.2) for the TWiST state, and −2.08 months (−2.6, −1.6) for the REL state. The mean duration of Q-TWiST was 21.07 months for zanubrutinib versus 18.67 months for ibrutinib. The estimated difference in means (95% CI) for Q-TWiST gain was significantly higher for zanubrutinib versus ibrutinib (2.40 months; 1.9, 2.9; P<0.001), and the relative Q-TWiST gain was 9.14% (Figure). In the sensitivity analysis, using the intent-to-treat population, the mean difference (95% CI) for Q-TWiST gain was 1.30 months (1.0, 1.6; P=0.05) for zanubrutinib versus ibrutinib, and the relative Q-TWiST gain was 4.63%. Conclusions: This Q-TWiST analysis demonstrated a statistically significant gain in quality-adjusted survival with zanubrutinib compared with ibrutinib in patients with high-risk R/R CLL. The slightly longer TOX duration in patients receiving zanubrutinib versus those receiving ibrutinib could be explained in part by better treatment adherence. The results of this Q-TWiST analysis, which integrates both the length and quality of survival in addition to efficacy and toxicity, provide valuable insights that may help to inform clinical decision-making in the treatment of patients with R/R CLL.

A Parallel Comparison of the Safety and Efficacy of Allogeneic Hematopoietic Stem Cell Transplantation for Refractory and Relapsed T-ALL/Lbl Patients Who Achieved Complete Remission with CD7 CAR-T Versus Patients Who Achieved First Complete Remission with Chemotherapy before Transplantation

Introduction We have developed a fratricide-resistant CD7 CAR-T cells from bulk T cells transduced with CD7 CAR without gene editing or additional gene transfer, named naturally selected CD7 CAR-T (NS7CAR-T). NS7 CAR-T therapy has shown a robust complete remission (CR) rate in patients with refractory or relapsed (R/R) T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma (T-ALL/LBL). Nonetheless, the risk of relapse remains a challenge post-CD7 CAR-T therapy alone, suggesting the potential benefits of consolidation allogeneic hematopoietic stem cell transplantation (allo-HSCT). The question remains whether CD7 CAR-T therapy could increase the incidence of post-transplant complications. Here we conduct a parallel study to compare the safety and efficacy of allo-HSCT in R/R T-ALL/LBL patients who achieved CR with NS7 CAR-T therapy vs those who reached first CR (CR1) using chemotherapy prior to transplantation. Methods Between November 2020 and January 2023, a total of 223 consecutive patients with T-ALL/LBL who underwent allo-HSCT at our center were retrospectively analyzed. Patients who met the following criteria were included in this parallel study: 1) underwent the first allo-HSCT in our hospital, 2) achieved CR before transplantation either from NS7CAR-T therapy or CR1 from chemotherapy (Fig. 1). A total of 139 patients met the criteria for analysis and underwent allo-HSCT after achieving CR either from NS7CAR-T therapy (n=43) or chemotherapy (CR1=96). The median patient age at the time of transplantation was 15 years (range: 1-51 years). The median follow-up time was 370 days (2-941 days). CD7 CAR-T patients were from two clinical trials registered at www.clinicaltrials.gov NCT04572308, NCT04916860. Transplant donor types included sibling identical (n=12), haploidentical (n=110), and unrelated donors (n=17). We used total body irradiation (TBI)-based conditioning regimens (n=128) or Busulfan/Melphalan-based regimens (n=11). To prevent graft-versus-host disease (GVHD), we used ATG along with a short-term regimen combining methotrexate, cyclosporine/tacrolimus, and mycophenolate mofetil. Results The characteristics of patients from both groups are detailed in Table 1. The median interval between CD7 CAR-T treatment and transplantation was 59 (39-131) days. Within the CAR-T group, 16 patients had primary refractory disease, 18 had relapsed disease and 9 in minimal residual disease (MRD)-positive status in bone marrow before CD7 CAR-T therapy. Prior to CAR-T therapy, 55.8% (24 patients) had extramedullary lesions. In comparing the CAR-T and chemotherapy CR1 groups, we observed no significant differences in long-term efficacy post-allo-HSCT. The 2-year leukemia-free survival (LFS) rates were 64.9 (95% CI, 49.1-79.2)% and 69.8 (95% CI, 59.0-79.6)% (p=0.340); the 2-year overall survival (OS) rates were 64.1 (95%CI, 48.0-78.6)% and 71.8 (95%CI, 60.5-81.9)% (p=0.107); the 2-year relapse incidence (RI) rates were 9.3 (95%CI, 3.7-23.7)% and 11.5 (95%CI, 6.4-20.7)% (p=0.873); and the 2-year non-relapsed mortality (NRM) rates were 28.3 (95% CI, 16.9-47.4)% and 21.6 (95% CI, 13.9-33.4)% (p=0.244), for the CD7 CAR-T and non-CAR-T groups, respectively. The incidence of all grades of acute GVHD (aGVHD) 37.2 (95%CI, 25.2-54.9)% vs. 42.1 (95%CI, 33.3-53.3)% did not significantly differ between the CAR-T and non-CAR-T groups. A trend toward an increased rate but no P significance was observed in Grade 3-4 aGVHD incidence of 20.93 (95%CI, 11.7-37.4)% vs. 9.5 (95%CI, 5.1-17.6)% (p=0.139). One-year incidences of moderate to severe chronic GVHD (cGVHD) were 12.2 (95%CI, 5.4-28.0)% and 18.0 (95%CI, 11.5-28.0)% (p=0.357) in the CD7CAR-T and non-CAR-T groups, respectively (Table 2). No statistically significant differences were detected in the occurrences of transplant-associated thrombotic microangiopathy (TA-TMA), CMV, EBV, and HHV-6 infections between the two groups. Upon analyzing, we found that r/r patients who received allo-HSCT after achieving remission with NS7 CAR-T had a similar OS to CR1 patients (Fig. 2). Conclusions Our parallel study showed that T-ALL/LBL patients, who achieved CR via CD7 CAR-T therapy followed by a consolidation allo-HSCT, had similar favorable OS and LFS compared to patients who underwent allo-HSCT in the CR1 state induced by chemotherapy. This was achieved without increasing the incidence of GVHD, infections, or TA-TMA.

Revumenib Maintenance Therapy Following Revumenib-Induced Remission and Transplant

Background: The potent and selective oral menin-histone-lysine N-methyltransferase 2A (KMT2A) inhibitor revumenib (SNDX-5613) induced complete remissions with clearance of residual disease in acute leukemias refractory to multiple previous lines of therapy ( Nature. 2023;615:920-924). Here we report outcomes following the resumption of revumenib after hematopoietic stem cell transplant (HSCT). Methods: Patients aged ≥30 days with relapsed or refractory (R/R) acute leukemias with genetic alterations associated with HOXA overexpression, including KMT2A-rearranged ( KMT2Ar) or nucleophosmin 1 ( NPM1)-mutated leukemias, were enrolled in the AUGMENT-101 phase 1/2, multicenter, open-label, dose-escalation study (NCT04065399). Patients received revumenib every 12 hours (q12h) in 28-day continuous cycles. Patients who achieved a complete response (composite definition [CRc]), morphological leukemia-free state, or partial response could undergo HSCT without leaving the study. Revumenib was stopped before the HSCT conditioning regimen but, per a protocol amendment, could be resumed after HSCT if the patient was in CRc. Measurable residual disease (MRD) was assessed using multiparameter flow cytometry and/or polymerase chain reaction. Results: Between November 5, 2019, and June 28, 2023, a total of 131 phase 1 patients were enrolled and treated in the AUGMENT-101 study. Nine patients with acute myeloid leukemia (AML) from the AUGMENT-101 study resumed revumenib (8 after transplant and 1 after a stem cell boost [Table 1]). Seven patients had KMT2Ar and 1 patient each had nucleoporin 98 rearranged (NUP98r) and mutated NPM1. Revumenib was resumed 59 to 180 days after HSCT (Table 2). Dose was reduced for 4 of 9 patients to mitigate adverse events of thrombocytopenia (n=3) and leg pain (n=1), and revumenib was discontinued in 4 of 9 patients because of progressive disease (n=2), cytopenias (n=1), and not reported (n=1). Revumenib duration of treatment (DOT) in the maintenance setting at the time of this analysis ranged from 23 to 588 days, with treatment ongoing for 5 of the 9 patients. CRc was maintained in 6 of 9 patients after HSCT and maintenance revumenib. One patient with reported MRD after HSCT converted to MRD-negative status following initiation of revumenib maintenance therapy. Overall, MRD-negative remissions were maintained in 5 patients as of the data cut off. Conclusions: With 3 patients on revumenib maintenance therapy for more than a year, long-term responses, including conversion to MRD-negative status, were seen in these heavily pretreated patients with AML. Resuming revumenib post HSCT had a tolerable safety profile consistent with that previously reported for the AUGMENT-101 study.

Efficacy and Safety of the Second CAR-T Therapy in Patients with Refractory/Relapsed Acute B-Cell Lymphoblastic Leukemia

Background: Chimeric antigen receptor T (CAR-T) cells therapy has shown significant efficacy in relapsed or refractory B-cell acute lymphoblastic leukemia (R/R B-ALL), but a substantial proportion of patients still relapse after a period of remission. The efficacy of a second CAR-T cell infusion is uncertain. The aim of this study was to explore the efficacy and safety of the second CAR-T therapy in R/R B-ALL patients. Methods: Between August 2018 and October 2022, 35 patients with R/R B-ALL successfully received the second CAR-T treatment. Among them, 18 patients received single CD19 or CD22 CAR-T therapy, 14 patients received tandem CD19/CD22 CAR-T therapy, and 3 patients received sequential CD19 and CD22 CAR-T therapy. Results: The complete remission (CR) rates were 55.6% (10/18) in patients who received CD19 or CD22 CAR-T therapy, 85.7% (12/14) in patients who received tandem CD19/CD22 CAR-T therapy, and 33.3% (1/3) in patients who received sequential CD19 and CD22 CAR-T therapy (tandem CD19/CD22 vs. others, P=0.045). Tandem CD19/CD22 CAR-T therapy remained one of the significant favorable factors in multivariate logistic regression analysis of CR rate in all patients (hazard ratio, 0.128; 95% CI, 0.017-0.974). A total of 5 patients with severe cytokine release syndrome (Grade ≥ 3) were observed, including 2 patients in the single CD19 CAR-T group, 2 patients in the tandem CD19/CD22 CAR-T group, and 1 patient in the sequential CD19 and CD22 CAR-T group. Of the 23 patients who achieved CR, 4 patients bridged to allogeneic hematopoietic stem cell transplantation (allo-HSCT) and 3 patients received decitabine (DAC) consolidation.With the median follow-up of 25.5 months (range, 1.1 to 33.5), the 2-year overall survival, leukemia-free survival (LFS) and cumulative incidence of relapse rates were 23.7%, 30.5% and 69.5%, respectively. Multivariate Cox regression analyses showed that a better LFS related to the absence of high-risk cytogenetics and genetic characteristics, DAC combination with fludarabine and cyclophosphamide lymphodepletion regimen, and bridging allo-HSCT or DAC consolidation. Conclusions: Our study showed that the second infusion of tandem CD19/CD22 CAR-T therapy obtains a better response than other infusion strategies. Bridging allo-HSCT or DAC consolidation had a significant survival benefit in patients with CR after the second CAR-T therapy.

Senza5 TM CART5: An Autologous CD5-Deleted Anti-CD5 CART Product with Enhanced Anti-T-Cell Lymphoma Activity

Introduction: Treatment with adoptive T cell therapies expressing Chimeric Antigen Receptors (CART) produces unprecedented clinical responses in relapsed or refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL), non-Hodgkin lymphomas (NHL), and multiple myeloma. Despite the transformative potential of these therapies for cancer, most patients treated with CART therapy experience incomplete responses or eventual relapse. Moreover, CART therapy is not yet effective for solid cancers or other hematological malignancies such as T-cell neoplasms. Thus, there is a dire need to enhance CART efficacy and extend this approach to more cancer types. We previously presented preliminary preclinical results of CD5-directed CAR-T cells (CART5) that had CD5 knocked out (KO) [ Blood (2020) 136 (Supplement 1): 51-52]. CD5 KO not only prevented CART fratricide but also dramatically enhanced CART function in multiple in vivo models. Enhanced CART function reflected disinhibition of early T-cell activation by abrogating CD5 signaling. Here, we combine the functional advantage of CD5 KO CART5 with a clinical-grade “rapid” 5-day manufacturing process, characterize the resulting drug product, and evaluate its in vivo antitumor efficacy in a human xenograft model of T-cell lymphoma/leukemia. Methods and Results: Using normal healthy donor T cells, we developed a proprietary, “rapid” 5-day GMP-validated manufacturing process for CD5 KO CART5, completing the process in 5 days (CART5 rapid) instead of the conventional 14+ days (CART5 conv). At the end of manufacturing, the two products possessed similar ratios of CD8 + and CD4 + T cells, CD5 editing, and CAR5 expression. The CART5 rapid product did not exhibit any genetic or functional abnormalities characteristic of tumorigenic risk, demonstrated by low off-target gene editing in low-risk genes, no in vitro soft agar colony formation, and normal karyotypes. Importantly, the CART5 rapid manufacturing process did not skew TCRβ profiles, indicating that the CART5 rapid product retains a diverse T-cell repertoire. CD5 KO CART5 rapid cells possessed high specificity for T-cell leukemia/lymphoma, reflected by the absence of recognition of ex vivo normal human cells (cardiomyocytes, hepatocytes, neurons, monocytes, etc). Interestingly, CD5 KO CART5 rapid cells possessed a more central memory-like phenotype than CD5 KO CART5 conv cells which has been correlated with improved clinical efficacy. To compare their antitumor efficacy, we performed an in vivo experiment with CD5 + Jurkat cells utilizing a suboptimal dose of CART5 cells. Immunodeficient NSG mice were engrafted with 10 6 Jurkat cells on day -7 and treated intravenously on day 0 with a suboptimal dose (0.5x10 6 CAR + cells) of CD5 KO CART5 rapid or CD5 KO CART5 conv cells. Controls received an equivalent total number of Mock KO UTD conv. Mice treated with CD5 KO CART5 rapid cells demonstrated superior antitumor responses compared to CD5 KO CART5 conv. Improved tumor control correlated with dramatically increased overall survival of mice treated with CD5 KO CART5 rapid (median overall survival Mock KO UTD = 21 days; CD5 KO CART5 conv = 44 days vs. CD5 KO CART5 rapid = not reached, p=0.0019, Mantel-Cox; Figure 1). Conclusion: This novel CD5 KO anti-CD5 CART product manufactured in a 5-day GMP process shows high specificity and significantly enhanced antitumor efficacy resulting from the synergistic effect of improved early T-cell activation due to CD5 deletion and preservation of T-cell memory phenotypes conferred by a novel, 5-day manufacturing processes. Autologous Senza5 TM CART5 for the treatment of CD5 + nodal T-cell lymphomas is expected to enter the clinic in a Phase I study later this year.

IL-10 Levels Differentially Regulate IFNg Signaling of CART19 and CART22

Introduction: CD19-directed CART (CART19) are highly effective in the treatment of relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). However, some pts relapse with CD19 negative disease after CART19, which has led to the development of CD22-directed CART (CART22). While CART22 has produced 70% CR in CD19 negative ALL, this is lower than CART19 and there is less evidence for long-term disease control compared to CART19. Interestingly, toxicity patterns are different with CART22 pts experiencing a later and less severe CRS even at high disease burden. To better understand these differences in efficacy and toxicity we conducted comprehensive serum proteomic profiling on serial samples from pts treated with CART-19 or -22. Methods: Serial serum samples from pts enrolled on two clinical trials of the murine CART19 CTL019 (NCT01626495 & NCT02906371) and CART22 (NCT02650414) were examined. Pts were categorized as having severe (Grade 3 or 4) or minimal CRS (Grade 1 or 2). All statistical analyses were performed using R (v4.2.2) and PRISM (v9). Differential expression analyses were performed between CART19 and CART22 pts. Significance was defined as an absolute fold change > 2, and a false discovery rate (FDR) < 0.05. All in vitro experiments were performed in triplicate. Two-tailed t tests were calculated. Results: 43 pts (CART19 N=26 and CART22 N=17) with 223 unique timepoints were included in this analysis. All CART22 pts had minimal CRS; 13 CART19 pts had minimal and 13 had severe CRS. Both cohorts had high disease burden prior to infusion (CART19 mean = 29.9%, CART22 mean=44.3%, p=0.19). All pts who received CART22 had previously received CD19 directed immunotherapy (CART19 N=16, blinatumomab N=1). At the pre-infusion timepoint, 12 proteins were significantly different between pts in the CART19 and CART22 cohorts. The protein most significantly differentially expressed in patients who subsequently underwent CART22 treatment was the anti-inflammatory cytokine IL-10. IL-10 levels were consistently elevated in patients who received CART22, surpassing levels at all observed timepoints, even when compared with patients treated with CART19 who developed severe CRS (See Fig 1). We therefore hypothesized the IL-10 levels may be impacting the efficacy and CRS associated with CART22. We next tested the impact of IL-10 on IFNγ as a surrogate marker of CRS in a series of in vitro experiments. CART-19 or -22 cells from the same donor were co-cultured with NALM-6 cells in the presence and absence of IL-10. Without IL-10 present, CART22 cells produced higher levels of IFNγ than CART19 cells (8959 vs 6395 pg/mL, p=0.005). Inversely, with IL-10 present, CART19 cells produced more IFNγ than CART22 cells (21006 vs 15157 pg/mL, p=0.05). We evaluated the length of immune synapse (IS) formation, the time between CART cell interaction and blast killing, using live-cell microscopy, and found that CART22 cells had a trend towards a prolonged IS formation relative to CART19 cells (median time to detachment 9.1 min vs. 3.5 min, p=ns). Finally, given the differences in time of onset of CRS following CART19 vs CART22, we looked at levels of IFNγ and its canonical responder CXCL9 between pts with minimal CRS only. As expected, we found a later peak of IFNγ in CART22 pts compared to CART19 pts (Figure 2). Interestingly, despite similar peaks in IFNγ, CXCL9 levels were higher in CART22 pts, implying dysregulation of the IFNγ/CXCL9 axes in CART22 but not CART19 pts. Conclusions: Comparing comprehensive serum proteomic profiling between CART19 and CART22 pts we make several novel observations. First, IL-10 levels are significantly higher in CART22 pts at pre-infusion, suggesting that previous immunotherapy has caused an upregulation of this anti-inflammatory cytokine. Second, we show that IL-10 levels have a differential impact on IFNγ levels in CART19 vs CART22 cells. This may impact both CRS associated with CART cells and efficacy and may contribute to observed differences in IFNγ and CXCL9 levels seen between the two patient populations. These findings are significant for the clinical translation of CART22 and other immunotherapies, as previous immunotherapy may lead to sustained alterations in the cytokine milieu. Furthermore, IL-10 is a targetable cytokine that could be modulated in future studies to improve efficacy of CART22.

Safety and Efficacy of Anakinra in Haemophagocytic Lymphohistiocytosis Associated with Acute Leukaemia

Haemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition in which immune hyperactivation and dysregulation results in a cytokine storm. It may be primary or secondary to infection, autoimmune disorders or malignancies (HLH-M). Lymphoma is the most widely described association in HLH-M; there is very limited data linking acute leukaemia and HLH. Current recommended treatments for HLH-M include steroids, IVIG and etoposide-containing regimens. In contrast, the recommended treatment in HLH- Macrophage activation syndrome is anti-IL1 directed therapy such as anakinra. In 2021 NHS England approved the use of anakinra to treat HLH of any cause, but the safety and efficacy data of anakinra in HLH-M is limited. We report a retrospective review of all patients diagnosed with HLH and acute leukaemia (AML) or acute lymphoblastic leukaemia (ALL) in a single London tertiary referral centre (University College London Hospitals NHS Foundation Trust (UCLH)), between 1 January2019 and 31 December 2022. We analysed electronic health records (EHR) of all patients diagnosed with ALL or AML and cross-referenced this with records of all patients referred to the UCLH HLH MDT. This identified 468 patients with acute leukaemia, of whom 13 (2.8%) were diagnosed with HLH. For diagnostic purposes, we utilised H scores (comprising laboratory and clinical parameters providing an HLH probability for each patient) using clinical and laboratory data collected from the EHR. HLH was diagnosed in patients with a clinical suspicion of HLH and an H score of >169. Patients were treated with various types of immunosuppressive therapy (IST). Twelve (92%) received anakinra, three (17%) of whom had anakinra monotherapy whilst the remainder had anakinra with steroids (38%), steroids and IVIG (15%), or steroids, IVIG and etoposide (8%). Anakinra was given intravenously in eleven patients and subcutaneously in one, with a mean daily dose of 4.6mg/kg. All patients required critical care input during their HLH treatment. Eight (62%) were admitted to intensive care unit (ICU) on diagnosis with HLH and required ventilatory and vasopressor support; the remainder were reviewed by critical care outreach. 92% patients had an initial rapid clinicopathological response following IST, becoming afebrile, reduction in oxygen requirement and haemodynamic stability seen within 24-72 hours. One patient (8%) had an initial clinical response to anakinra, methylprednisolone and IVIG triple therapy, but displayed subsequent rapid deterioration in the context of pseudomonal infection and died of multiorgan failure. Nine patients (69%) had no clinicopathological recurrence of HLH following initial treatment. Three (23%) had an initial clinical response to IST but had subsequent biochemical and clinical relapse of HLH. These patients all had relapsed/refractory disease on HLH recurrence. Only one (8%) patient died from HLH. Eight (62%) remain in remission from leukaemia at the end of the study period. Three patients (23%) died of refractory leukaemia, whilst a fourth patient is receiving palliative care for refractory disease. Although the link between HLH and lymphoma is well characterised, there is limited data on HLH in the context of acute leukaemia. In this case series, 2.8% of the patients treated with acute leukaemia developed HLH, which is comparable to the 3% for lymphoma quoted elsewhere. Although 92% of patients had microbiological or radiological evidence of infection on HLH diagnosis, many infections involved organisms not classically associated with HLH e.g. rhinovirus, indicating that the underlying leukaemia may play a central role in HLH-M pathogenesis. This is highlighted by the relapse of HLH which occurred alongside leukaemia relapse in three patients. Treatment responses and outcomes were favourable compared to the existing literature. Tamamyan et al reported an overall survival rate of 15% in 13 patients with HLH-M. Delavigne et al reported a three-month mortality rate of 36% in AML patients presenting with HLH during induction, compared to our figure of 23% for this time period and our overall survival rate of 62%. We believe this is the first case series demonstrating the effectiveness and high overall survival of HLH-M patients with concurrent leukaemia diagnosis using anakinra. Early treatment with anakinra alongside other immunosuppression may explain the trend towards improved outcomes in our cohort.

Efficacy and Safety of Asciminib in Chinese Patients with Philadelphia Chromosome(Ph)-Positive Leukaemias

Background Asciminib is a first-in-class STAMP (Specifically Targeting the ABL Myristoyl Pocket) tyrosine kinase inhibitor (TKI). Its efficacy and safety profile in patients with chronic-phase chronic myeloid leukaemia (CML) have been shown in the Phase 3 ASCEMBL study, comparing asciminib 40 mg twice daily versus bosutinib 500 mg once daily. However, asciminib use in Chinese patients has hitherto not been reported. Moreover, the safety of high-dose asciminib (≥150 mg twice daily) in the Asian population is yet to be addressed. Here we present the real-world clinical outcomes of Chinese patients with Ph+ leukaemias in Hong Kong who received asciminib through a Managed Access Program (MAP). Methods Queen Mary Hospital (QMH) is a university-affiliated tertiary hospital in Hong Kong. Patients with Ph+ leukaemias under the care of QMH for whom asciminib was requested through MAP were included. Details on patient demographics, disease history, prior treatment lines, asciminib usage, adverse events, efficacy and death were collected. Results At data cut-off for analysis on 30 June 2023, 13 patients for whom asciminib was approved had data available. One patient passed away before the commencement of asciminib, and thus was excluded (Table 1). There were 7 patients with CML, who on diagnosis were all in chronic phase at a median age of 53 (22-70) years. All had received imatinib and at least one newer generation TKI, with 5 patients (71%) having received ponatinib. The majority (5 patients, 71%) received asciminib because of lack of efficacy of prior treatment, with 4 patients also having the T315I mutation. The median age at asciminib treatment (median dose: 400 mg, 80-400 mg) was 64 (28-76) years, with a median time from diagnosis of 81 (13-314) months. Two patients received imatinib concomitantly. The median duration of treatment was 12 (6-38) months. Two patients were already in at least major molecular response (MMR) before treatment, and have maintained that response. Two patients had ≥ 1-log reduction of BCR::ABL1 transcript level. Three patients had stable disease. There was one death unrelated to CML (Figure 1). There were 5 patients with Ph-positive acute lymphoblastic leukaemia (ALL), at a median age of 57 (18-68) years at diagnosis. All had received prior chemotherapy in combination with a TKI (imatinib, N=3; dasatinib, N=5; ponatinib, N=5), with 3 patients having undergone allogeneic haematopoietic stem cell transplantation (allo-HSCT). Three patients had detectable kinase domain mutation (T315I, N=2; E255K + E459K, N=1). The majority (3 patients, 60%) received asciminib because of lack of efficacy of prior treatment. The median age at asciminib treatment (median dose: 240 mg, 80-400 mg) was 58 (19-72) years, with a median time from diagnosis of 22 (7-47) months. The median duration of treatment was 16 (2-19) months. Four patients received another ATP-competitive TKI concomitantly (imatinib, N=1; dasatinib, N=1; ponatinib, N=2). For patients receiving asciminib due to prior treatment failure (N=3), complete molecular response (CMR) was achieved in 2 patients with measurable residual BCR:: ABL1; 1 patient with frank leukaemia achieved complete remission with incomplete hematologic recovery with molecular response at 0.06%. For two patients receiving asciminib because of intolerance to prior treatment, one further improved from MR4 to CMR while pre-existing CMR was maintained in the other patient. There were 3 deaths; 2 due to COVID-19 and 1 due to refractory leukaemia (Figure 1). Asciminib showed an acceptable safety profile, with raised lipase leading to dose reduction only in 1 patient. Grade 3 or above toxicities were all haematological, observed in 5 patients, 2 of whom had previous allo-HSCT. One patient on asciminib-ponatinib treatment developed grade 2 renal impairment. Conclusion Asciminib demonstrated promising clinical efficacy with satisfactory tolerance in this cohort of Ph+ leukaemias failing multiple lines of therapy. Further investigation of its therapeutic role in Ph+ ALL is warranted.